RESUMO
ARP4, an essential gene of Saccharomyces cerevisiae, codes for a nuclear actin-related protein. Arp4 is a subunit of several chromatin-modifying complexes and is known to be involved in the transcriptional regulation in yeast. We used a mutant strain with a single amino acid substitution (G161D) in the conserved actin fold domain to investigate the influence of Arp4 on stress and nitrogen catabolite repression genes. The deficiency of functional Arp4 caused a highly increased sensitivity towards nitrogen starvation and to the macrolide antibiotic rapamycin. We show the changes of mRNA levels of selected genes under these conditions. The upregulation of stress genes as a consequence of treatment with rapamycin was largely Msn2p/Msn4p-dependent. The sensitivity towards rapamycin indicates a participation of Arp4 in the regulation of the TOR pathway. Consistently, arp4G161D cells exhibited an affected cell cycle. Long-term cultivation, which leads to a G1 arrest in wild-type cells, provoked arrest in G2/M (more than 60%) in the mutant strain. The same effect was observed upon treatment with rapamycin, indicating an unexpected relationship of Arp4 to TOR-mediated cell cycle arrest.
Assuntos
Actinas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Actinas/genética , Actinas/fisiologia , Antifúngicos/farmacologia , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Supressores , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Mutação Puntual/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Sirolimo/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
A mutational analysis of the essential nuclear actin-related protein of Saccharomyces cerevisiae, Act3p/Arp4p, was performed. The five residues chosen for substitution were amino acids conserved between actin and Act3p/Arp4p, the tertiary structure of which most probably resembles that of actin. Two thermosensitive (ts) mutants, a single and a double point mutant, and one lethal double point mutant were obtained. Both ts mutants were formamide-sensitive which supports a structural relatedness of Act3p/Arp4p to actin; they were also hypersensitive against hydroxyurea and ultraviolet irradiation pointing to a possible role of Act3p/Arp4p in DNA replication and repair. Their 'suppressor of Ty' (SPT) phenotype, observed with another ts mutant of Act3p/Arp4p before, suggested involvement of Act3p/Arp4p in transcription regulation. Accordingly, genome-wide expression profiling revealed misregulated transcription in a ts mutant of a number of genes, among which increased expression of various stress-responsive genes (many of them requiring Msn2p/Msn4p for induction) was the most salient result. This provides an explanation for the mutant's enhanced resistance to severe thermal and oxidative stress. Thus, Act3p/Arp4p takes an important part in the repression of stress-induced genes under non-stress conditions.
